臺灣博碩士論文加值系統

摘 要
Rutamarin alcohol (RA) 是由芸香科植物芸香 (Ruta graveolens L.) 經分離純化之dihydrofuranocoumarin類化合物，我們進行RA之降血壓作用及血管舒張的機轉探討。在麻醉之Sprague-Dawley大白鼠，RA (0.25 - 1.25 mg/kg) 產生劑量相關性的降血壓作用。 在離體大白鼠胸主動脈含有完整內皮及去內皮細胞之血管環，RA (10-7 ~ 10-4 M) 對phenylephrine (PE, 10-6 M) 或KCl (60 mM) 所引起之血管收縮均具有舒張的作用，並且以含有完整內皮細胞的血管環舒張作用較明顯。 前處理RA (10-6 ~ 5X10-5 M)15分鐘後，產生濃度相關性之非競爭性抑制PE (10-9 ~10-5 M) 的收縮作用。 在無鈣的Kerbs’溶液中，RA (3X10-5, 5X10-5 M) 明顯降低phorbol 12-myristate 13-acetate (protein kinase C activator, 10-6 M) 所產生的持續性收縮，亦可明顯抑制norepinephrine (NE, 10-6 M) 在平滑肌細胞引起的內鈣釋出，以及濃度相關性抑制NE，KCl或PMA處理後的外鈣流入所引起的收縮現象。 前處理N-nitro-L-arginine (nitric oxide synthase inhibitor, 10-4 M, 15分鐘)、methylene blue (guanylate cyclase inhibitor, 5X10-5 M, 5分鐘)、1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (specific soluble guanylate cyclase inhibitor, 10-6 M, 15分鐘) 造成RA舒張作用曲線右移；而前處理tetraethylammonium chloride (Ca2+-activated potassium channel blocker, 10-6 M, 60分鐘) 不改變RA的舒張作用。 另外，對培養的血管內皮細胞，處理RA (10-4M,30分鐘) 明顯增加一氧化氮 (nitric oxide；NO) 釋放。 在接受器結合實驗中，RA對a1-腎上腺性接受器並無作用，對L-型鈣離子通道的dihydropyrindine結合位置有較弱的結合作用。 培養的血管平滑肌細胞，處理RA (10-6 ~ 10-4M) 1小時或5X10-5M處理 (1 ~ 24小時)，均不會降低平滑肌細胞的存活率。 綜合以上結果，我們認為RA造成血管環舒張的作用，主要是作用在平滑肌細胞，經由減少細胞內鈣的釋放、抑制PKC的作用以及減少細胞外鈣經由接受器開啟型鈣離子通道 (receptor-operated calcium channels) 及膜電位依賴型鈣離子通道 (voltage-dependent calcium channels) 流入細胞內；RA亦有少部分作用是經由促進內皮細胞釋放NO，產生血管舒張。RA產生血管舒張的作用可能是其引起血壓下降的原因之一。

Abstract
We have examined both the hypotensive effect and the mechanisms of vasorelaxation of rutamarin alcohol (RA), a dihydrofuranocoumarins compound isolated from Ruta graveolens L. of Rutaceae. Intravenous bolus injections of RA (0.25-1.25 mg/kg) in anesthetized Sprage-Dawley rats produced dose-dependent hypotensive effects. In isolated rat thoracic aorta rings with or without endothelium, RA (10-7 ~ 10-4 M) relaxed phenylephrine (PE, 10-6M)- or KCl (60 mM)-induced vasocontractions. The vasorelaxing effect was greater for intact than for endothelium-denuded tissues. Pretreatment with RA (10-6 ~ 5X10-5 M) for 15 min shifted the concentration-response curves for PE (10-9 ~ 10-5M) to the right and reduced the maximal responses. Ca2+-free Krebs’ solution RA (3X10-5 ~5X10-5M) suppressed the sustained contraction stimulated by phorbol 12-myristate 13-acetate (PMA, protein kinase C activator, 10-6 M). RA (3X10-5, 5X10-5 M) also attenuated the norepinephrine (NE, 10-6M)-induced contraction in Ca2+-free solution. In Ca2+-depleted, NE-, PMA- or high K+-treated aortic rings, preincubation with RA attenuated the Ca2+-induced contraction in a concentration-dependent fashion. In RA caused a rightward shift of the vasorelaxant response curve in endothelium-intact rings by N-nitro-L-arginine (nitric oxide synthase inhibitor, 10-4M, 15 min), methylene blue (guanylyl cyclase inhibitor, 5X10-5M, 5 min), and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, specific soluble guanylyl cyclase inhibitor, 10-6M, 15 min) pretreatment. Tetraethylammonium chloride (Ca2+-activated potassium channel blocker, 10-6M, 60 min) pretreatment did not reverse the vasorelaxation of RA. Additionally, RA (10-4M, 30 min) significantly increased nitric oxide (NO) release in cultured endothelial cells. Receptor binding assay indicated that RA competed with the dihydropyridine (DHP) ligand with a Ki value of 111±54 microM. However, RA did not interact with rat heart a1-adrenoceptors. In cultured vascular smooth muscle cells, the cell viability was not affected treatment with RA (10-6 ~10-4M) for 1 hr or 5X10-5 M for 1 to 24 hr. Taker together, the present results suggested that the vasorelaxing effect of RA was mediated mainly through inhibiting PKC, Ca2+ release and Ca2+ influx, through receptor-operated Ca2+ channels and voltage-dependent Ca2+ channels in vascular smooth muscle. Although the contribution seemed to be minor in nature, increase in NO release in endothelial cells, was also involved. The regulation of vascular smooth muscle cells and endothelial cells acts simultaneously to cause vasorelaxarion which could account, at least a part, for the hypotensive action.

誌謝
目錄
縮寫表..................................................... 1
中文摘要................................................... 3
英文摘要................................................... 5
壹、緒論....................................................7
1-2 研究背景..............................................7
1-1 研究動機...............................................13
貳、實驗方式...............................................16
2-1 麻醉大白鼠血壓、心跳測定實驗...........................16
2-2 離體大白鼠胸主動脈血管環張力實驗.......................17
2-3 內皮細胞產生NO的測定實驗...............................21
2-4 接受器結合實驗.........................................22
2-5 細胞存活率測定實驗.....................................24
2-6 統計方法...............................................25
參、實驗結果...............................................26
3-1 RA對大白鼠血壓及心跳的作用.............................26
3-2 RA對離體大白鼠胸主動脈血管環張力的作用.................26
3-3 RA對內皮細胞產生NO的作用...............................32
3-4 RA對接受器的結合作用...................................32
3-5 RA對平滑肌細胞存活率的作用.............................33
肆、討論...................................................34
伍、參考文獻...............................................43
陸、圖表...................................................50

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